Triterpenoids-templated self-assembly nanosystem for biomimetic delivery of CRISPR/Cas9 based on the synergy of TLR-2 and ICB to enhance HCC immunotherapy

Combination immunotherapy has shown promising potential for enhancing the objective response rate compared to immune checkpoint blockade (ICB) monotherapy. However, combination therapy with multi-drugs is limited by the different properties of the agents and inconsistent synergistic targeted delivery. Herein, based on a universal triterpene template and the anticancer active agent ursolic acid (UA), a cytomembrane-coated biomimetic delivery nanoplatform (UR@M) prepared by the self-assembly of a PD-L1 targeted CRISPR/Cas9 system and UA was designed for hepatocellular carcinoma (HCC) treatment. UR@M showed enhanced tumor accumulation in vivo with homologous tumor targeting, and CRISPR in the nanosystem exhibited potent gene-editing efficiency of 76.53% in vitro and 62.42% in vivo with no off-target effects. UA activated the natural immune system through the TLR-2-MyD88-TRAF6 pathway, which synergistically enhanced the proliferation of natural killer cells and dendritic cells and realized excellent immune cytotoxic T cell infiltration by combining with the ICB of PD-L1. The strategy of work along both lines based on innate immune and adaptive immunity displayed a significant effect in tumor regression. Overall, the UA-templated strategy “killed three birds with one stone” by establishing a self-assembly nanosystem, inducing tumor cell death, and promoting synergistic immunostimulation for HCC treatment.


Figure S5 .
Figure S5.Evaluation of the stability of nanodrugs in a physiological environment.The size particles were detected 14 days after the UA NPs, UR NPs and UR@M NPs dissolved in different solutions compared to Day 0. Data are presented as mean±SD (n=3).

Figure S7 .
Figure S7.The drug (UA, Cas9) release of different pH values on UR NPs in 72 h.Data are presented as mean±SD (n=3).

Figure S8 .
Figure S8.With the free ICG as the control, the fluorescence curve of UR and UR@M after the binding of ICG.

Figure S10 .
Figure S10.The quantification of UA in HepG2 cells at 2, 6, and 12 h after treated with UR@M NPs compared to the UA NPs and UR NPs.Data are presented as mean±SD (n=3).

Figure S11 .
Figure S11.Cellular uptake of UR@M NPs in H22 cells.(A).Fluorescent images of cells after incubation with UA NPs, UR NPs, and UR@M NPs for 6 h.(B).The fluorescence intensity detection of cells by flow cytometry.Scale bar= 20 μm.

Figure S12 . 17 Figure S13 .
Figure S12.Combination index (CI) of CRISPR system and drug co-delivery nanosystem (UR NPs) compared to the single Cas9/RNP or UA when treated with HepG2 cells.CI values < 1 indicated synergism, CI values = 1 indicated an additive effect, and CI values > 1 indicated antagonism.Data are presented as mean±SD (n=3).

Figure S14 .
Figure S14.The normal cells of L02, HEK-293T and HUVEC were treated with different drugs with various concentrations.Cell viability was measured by CCK-8 assay.Data are presented as mean±SD (n=3).

Figure S16 . 21 Figure S17 .
Figure S16.The weight of excised tumor was detected after the in vivo experiment.PBS group was treated as the control for significance analysis.Data are presented as mean±SD (n=3).* P < 0.05, ** P < 0.01, *** P < 0.001.

Figure S18 .
Figure S18.Body weight of tumor-bearing mice post the indicated treatment during the 21 days (n=6).

Figure S19 .
Figure S19.The weight of HCC orthotopic tumor mice and the pictures of liver after the treatment.

Figure S20 .
Figure S20.Hemolytic test of UA NPs, UR NPs, and UR@M NPs.The water and PBS were used as the positive (hemolysis ratio: 100 %) and negative (hemolysis ratio: 0 %) control.It was considered a hemolysis effect when the hemolysis ratio was more than 5 %.Data are presented as mean±SD (n=3).

Figure S21 . 26 Figure S22 .
Figure S21.The fluorescence ratio of tumor/liver ratio of UR NPs, UR NPs and UR@M NPs with the ICG group as the control.Data are presented as mean±SD (n=3).*** P < 0.001.

Table S2 .
The optimization of UR NPs.Data are presented as mean±SD (n=3).